Neurotransmitter transporter SC6

ABSTRACT

The SC6 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing SC6 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

FIELD OF THE INVENTION

[0001] This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides, to their use in therapy andin identifying compounds which may be agonists, antagonists and /orinhibitors which are potentially useful in therapy, and to production ofsuch polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

[0002] The drug discovery process is currently undergoing a fundamentalrevolution as it embraces ‘functional genomics’, that is, highthroughput genome- or gene-based biology. This approach as a means toidentify genes and gene products as therapeutic targets is rapidlysuperceding earlier approaches based on ‘positional cloning’. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

[0003] Functional genomics relies heavily on high-throughput DNAsequencing and the various tools of bioformatics to identify genesequences of potential interest from the many molecular biologydatabases now available. There is a continuing need to identify andcharacterise further genes and their related polypeptides/proteins, astargets for drug discovery.

[0004] In the central and peripheral nervous system, reliableneurotransmission depends on rapid termination of transmitter actionfollowing postsynaptic activation. In some cases this is achieved bymetabolism of the neurotransmitter, as in the case of acetycholine andneuropeptides. In many cases, however, including catecholamines,serotonin and some amino acids (e.g. GABA, glycine and glutamate), theneurotransmitter is efficiently removed into the presynaptic terminal orsurrounding glial cells by neurotransmitter transporters, membrane-boundpolypeptides located in the plasma membrane.

[0005] Recently, cDNAs encoding a number of Na/Cl-dependentneurotransmitter transporters (e.g. for serotonin, catecholamine, aminoacid (glycine, GABA)) have been described. The general structure of thisclass of transporter is very similar, containing twelve potentialtransmembrane helices and an external loop with 34 glycosylation sitesbetween transmembrane segments 3 and 4. In GABA and catecholaminetransporter subfamilies, the amino acid sequence is about 60-80%identical to other members within a subfamily and about 40% identical tomembers between two subfamilies (Liu et al., Proc. Natl. Acad. Sci. USA,(1992), 89:6639-6643). Transporters of amino acids such as glycine shareabout 40-50% homology with all members of the neurotransmittertransporter superfamily. Two classes of glycine transporter, GlyT-1 andGlyT-2, have been identified (Liu et al, J. Biol. Chem. (1992),268,22802-22808). Rat GlyT-2 has about 50% amino acid sequence identity witheither human or rat GlyT-1.

[0006] Glycine is a major transmitter in the nervous system. Glycine canhave both inhibitory and excitatory functions, which are mediated by twodifferent types of receptor, each associated with a different class ofglycine transporter. The excitatory function of glycine is mediated by“strychnine-insensitive” glycine receptors, which are part of the NMDAreceptor complex which mediates some of the actions of glutamate, themajor excitatory transmitter in the central nervous system. This type ofglycine receptor is widely distributed throughout the brain, and isassociated with the GlyT-1 transporter. Conversly, the inhibitory actionof glycine is mediated by “strychnine-sensitive” glycine receptors.These receptors are found mainly in the spinal cord, brainstem andcerebellum, and are associated with the GlyT-2 transporter.

[0007] Modulation of neurotransmitter transport enables synaptictransmission to be increased or decreased by altering the levels ofneurotransmitter in the synaptic cleft, and blockade of transport is anestablished approach to the treatment of psychiatric and neurologicalillness. Drugs which act by this mechanism include the tricyclicantidepressants, which act on monamine transporters in general, and theselective serotonin uptake inhibitors (SSRIs) (Lesch K P and Bengel D,CNS Drugs 4(1995), 302-322). A GABA transport inhibitor, tiagabine, hasrecently been identified as a potential therapy for epilepsy (Lesch K Pand Bengel D, CNS Drugs 4(1995), 302-322. Compounds which modulateglycine transporter function would be expected to alter synaptic levelsof glycine and thus affect receptor function. In the case of the GlyT-2transporter, inhibition of transporter function would produce increasedactivation of strychnine-sensitive glycine receptors. In the spinalcord, activation of these receptors would be expected to reducetransmission of pain-related information, so inhibition of the GlyT-2transporter could alleviate neuropathic or other pain sensation (e.g.Simpson R K et al, Neurochem. Res. (1996) 21, 1221-1226). In addition,activation of strychnine-sensitive glycine receptors can reduce musclehyperactivity, which can be related to conditions such as myoclonus,epilepsy and spasticity (e.g. Simpson R K et al, J. Spinal Cord Med.(1996) 19,215-224). Therefore, inhibition of the glycine transportercould alleviate spasticity or other muscle hyperactivity associated withepilepsy, stroke, head trauma, spinal cord injury, dystonia, multiplesclerosis amyotrophic lateral sclerosis, Huntington's Disease orParkinson's Disease

SUMMARY OF THE INVENTION

[0008] The present invention relates to SC6, in particular SC6polypeptides and SC6 polynucleotides, recombinant materials and methodsfor their production. In another aspect, the invention relates tomethods for using such polypeptides and polynucleotides, including thetreatment of neuropathic pain, other pain, spasticity, myoclonus,epilepsy, stroke, head trauma, spinal cord injury, dystonia, multiplesclerosis, amyotrophic lateral sclerosis, Huntington's Disease orParkinson's Disease, hereinafter referred to as “the Diseases”, amongstothers. In a further aspect, the invention relates to methods foridentifying agonists and antagonists/inhibitors using the materialsprovided by the invention, and treating conditions associated with SC6imbalance with the identified compounds. In a still further aspect, theinvention relates to diagnostic assays for detecting diseases associatedwith inappropriate SC6 activity or levels.

DESCRIPTION OF THE INVENTION

[0009] In a first aspect, the present invention relates to SC6polypeptides. Such peptides include isolated polypetides comprising anamino acid sequence which has at least 97% identity, preferably at least99% identity, to that of SEQ ID NO:2 over the entire length of SEQ IDNO:2. Such polypeptides include those comprising the amino acid of SEQID NO:2.

[0010] Further peptides of the present invention include isolatedpolypeptides in which the amino acid sequence has at least 97% identity,preferably at least 99% identity, to the amino acid sequence of SEQ IDNO:2 over the entire length of SEQ ID NO:2. Such polypeptides includethe polypeptide of SEQ ID NO:2.

[0011] Further peptides of the present invention include isolatedpolypeptides encoded by a polynucleotide comprising the sequencecontained in SEQ ID NO:1.

[0012] Polypeptides of the present invention are believed to be membersof the Neurotransmitter transporter family of polypeptides. They aretherefore of interest because of the established, proven history ofneurotransmitter transporters as therapeutic targets for the treatmentof neurological or psychiatric diseases. In particular, glycinetransporters would be expected to modulate activation of glycinereceptors by glycine, and there is evidence that strychnine-sensitiveglycine receptors can regulate transmission of pain information andmodulate muscle activity. Therefore, a therapeutic target for treatmentof diseases associated with altered muscle activity or pain transmissioncould be provided by a polypeptide which has the properties of a glycinetransporter, in particular of the GlyT-2 type which is associated withstrychnine-sensitive glycine receptors . . . These properties arehereinafter referred to as “SC6 activity” or “SC6 polypeptide activity”or “biological activity of SC6”. Also included amongst these activitiesare antigenic and immunogenic activities of said SC6 polypeptides, inparticular the antigenic and immunogenic activities of the polypeptideof SEQ ID NO:2. Preferably, a polypeptide of the present inventionexhibits at least one biological activity of SC6.

[0013] The polypeptides of the present invention may be in the form ofthe “mature” protein or may be a part of a larger protein such as aprecursor or a fusion protein. It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

[0014] The present invention also includes variants of theaforementioned polypeptides, that is polypeptides that vary from thereferents by conservative amino acid substitutions, whereby a residue issubstituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacids are substituted, deleted, or added in any combination.

[0015] Polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0016] In a further aspect, the present invention relates to SC6polynucleotides. Such polynucleotides include isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide which has atleast 97% identity, to the amino acid sequence of SEQ ID NO:2, over theentire length of SEQ ID NO:2. In this regard, polypeptides which have atleast 99% identity are highly preferred. Such polynucleotides include apolynucleotide comprising the nucleotide sequence contained in SEQ IDNO:1 encoding the polypeptide of SEQ ID NO:2.

[0017] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 90%identity, preferably at least 95% identity, to a nucleotide sequenceencoding a polypeptide of SEQ ID NO:2, over the entire coding region. Inthis regard, polynucleotides which have at least 97% identity are highlypreferred, whilst those with at least 98-99% identity are more highlypreferred, and those with at least 99% identity are most highlypreferred.

[0018] Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 90%identity, preferably at least 95% identity, to SEQ ID NO:1 over theentire length of SEQ ID NO:1. In this regard, polynucleotides which haveat least 97% identity are highly preferred, whilst those with at least98-99% identiy are more highly preferred, and those with at least 99%identity are most highly preferred. Such polynucleotides include apolynucleotide comprising the polynucleotide of SEQ ID NO:1 as well asthe polynucleotide of SEQ ID NO:1.

[0019] The invention also provides polynucleotides which arecomplementary to all the above described polynucleotides.

[0020] The nucleotide sequence of SEQ ID NO:1 shows homology with RatGlyT-2 glycine transporter (Q. R. Liu et al, J. Biol. Chem. (1993) 268:22802-22808) (GenBank L21672). The nucleotide sequence of SEQ ID NO:1 isa cDNA sequence and comprises a polypeptide encoding sequence(nucleotide 256 to 2649) encoding a polypeptide of 797 amino acids, thepolypeptide of SEQ ID NO:2. The nucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may be identical to the polypeptide encodingsequence contained in SEQ ID NO:1 or it may be a sequence other than theone contained in SEQ ID NO:1, which, as a result of the redundancy(degeneracy) of the genetic code, also encodes the polypeptide of SEQ IDNO:2. The polypeptide of the SEQ ID NO:2 is structurally related toother proteins of the Neurotransmitter transporter family, havinghomology and/or structural similarity with Rat GlyT-2 glycinetransporter (Q. R. Liu et al, J. Biol. Chem. (1993) 268: 22802-22808)(PIR A48716).

[0021] Preferred polypeptides and polynucleotides of the presentinvention are expected to have, inter alia, similar biologicalfunctions/properties to their homologous polypeptides andpolynucleotides. Furthermore, preferred polypeptides and polynucleotidesof the present invention have at least one SC6 activity. Polynucleotidesof the present invention may be obtained, using standard cloning andscreening techniques, from a cDNA library derived from mRNA in cells ofhuman spinal cord, using the expressed sequence tag (EST) analysis(Adams, M. D., et al. Science (1991) 252:1651-1656; Adams, M. D. et al.,Nature, (1992) 355:632-634; Adams, M. D., et al., Nature (1995) 377Supp:3-174). Polynucleotides of the invention can also be obtained fromnatural sources such as genomic DNA libraries or can be synthesizedusing well known and commercially available techniques.

[0022] When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself; or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, amarker sequence which facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

[0023] Further embodiments of the present invention includepolynucleotides encoding polypeptide variants which comprise the aminoacid sequence of SEQ ID NO:2 and in which several, for instance from 5to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted,deleted or added, in any combination.

[0024] Polynucleotides which are identical or sufficiently identical toa nucleotide sequence contained in SEQ ID NO:1, may be used ashybridization probes for cDNA and genomic DNA or as primers for anucleic acid amplification (PCR) reaction, to isolate full-length cDNAsand genomic clones encoding polypeptides of the present invention and toisolate cDNA and genomic clones of other genes (including genes encodingparalogs from human sources and orthologs and paralogs from speciesother than human) that have a high sequence similarity to SEQ ID NO:1.Typically these nucleotide sequences are 70% identical, preferably 80%identical, more preferably 90% identical, most preferably 95% identicalto that of the referent. The probes or primers will generally compriseat least 15 nucleotides, preferably, at least 30 nucleotides and mayhave at least 50 nucleotides. Particularly preferred probes will havebetween 30 and 50 nucleotides. Particularly preferred primers will havebetween 20 and 25 nucleotides.

[0025] A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess which comprises the steps of screening an appropriate libraryunder stringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof; and isolating full-lengthcDNA and genomic clones containing said polynucleotide sequence. Suchhybridization techniques are well known to the skilled artisan.Preferred stringent hybridization conditions include overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured, sheared salmon sperm DNA; followed by washing the filters in0.1×SSC at about 65° C. Thus the present invention also includespolynucleotides obtainable by screening an appropriate library understingent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof.

[0026] The skilled artisan will appreciate that, in many cases, anisolated cDNA sequence will be incomplete, in that the region coding forthe polypeptide is short at the 5′ end of the cDNA. This is aconsequence of reverse transcriptase, an enzyme with inherently low‘processivity’ (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during 1st strand cDNAsynthesis.

[0027] There are several methods available and well known to thoseskilled in the art to obtain full-length cDNAs, or extend short cDNAs,for example those based on the method of Rapid Amplification of cDNAends (RACE) (see, for example, Frohman et al., PNAS USA 85, 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the ‘missing’ 5′ endof the cDNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using‘nested’ primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the known gene sequence). The products of thisreaction can then be analysed by DNA sequencing and a full-length cDNAconstructed either by joining the product directly to the existing cDNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0028] Recombinant polypeptides of the present invention may be preparedby processes well known in the art from genetically engineered hostcells comprising expression systems. Accordingly, in a further aspect,the present invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

[0029] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis et al., Basic Methods inMolecular Biology (1986) and Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). Preferred such methods include, forinstance, calcium phosphate transfection, DEAE-dextran mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

[0030] Representative examples of appropriate hosts include bacterialcells, such as Streptococci, Staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 andBowes melanoma cells; and plant cells.

[0031] A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0032] If a polypeptide of the present invention is to be expressed foruse in screening assays, it is generally preferred that the polypeptidebe produced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

[0033] Polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography is employed forpurification. Well known techniques for refolding proteins may beemployed to regenerate active conformation when the polypeptide isdenatured during intracellular synthesis, isolation and or purification.

[0034] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofthe gene characterised by the polynucleotide of SEQ ID NO:1 which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredspatial or temporal expression of the gene. Individuals carryingmutations in the gene may be detected at the DNA level by a variety oftechniques.

[0035] Nucleic acids for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniquesprior to analysis. RNA or cDNA may also be used in similar fashion.Deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labeled SC6 nucleotidesequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing (ee, e.g.,Myers et al., Science (1985) 230:1242). Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method (see Cotton etal., Proc Natl Acad Sci USA (1985) 85:4397-4401). In another embodiment,an array of oligonucleotides probes comprising SC6 nucleotide sequenceor fragments thereof can be constructed to conduct efficient screeningof e.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability (see for example: M. Chee et al.,Science, Vol 274, pp 610-613 (1996)).

[0036] The diagnostic assays offer a process for diagnosing ordetermining a susceptibility to the Diseases through detection ofmutation in the SC6 gene by the methods described. In addition, suchdiseases may be diagnosed by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof polypeptide or mRNA. Decreased or increased expression can bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides, such as, for example, nucleicacid amplification, for instance PCR, RT-PCR, RNase protection, Northernblotting and other hybridization methods. Assay techniques that can beused to determine levels of a protein, such as a polypeptide of thepresent invention, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

[0037] Thus in another aspect, the present invention relates to adiagnostic kit which comprises:

[0038] (a) a polynucleotide of the present invention, preferably thenucleotide sequence of SEQ ID NO: 1, or a fragment thereof;

[0039] (b) a nucleotide sequence complementary to that of (a);

[0040] (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2 or a fragment thereof; or

[0041] (d) an antibody to a polypeptide of the present invention,preferably to the polypeptide of SEQ ID NO:2.

[0042] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component. Such a kit will be of use indiagnosing a disease or suspectability to a disease, particularlyneuropathic pain, other pain, spasticity, myoclonus, epilepsy, stroke,head trauma, spinal cord injury, dystonia, multiple sclerosis,amyotrophic lateral sclerosis, Huntington's Disease or Parkinson'sDisease, amongst others.

[0043] The nucleotide sequences of the present invention are alsovaluable for chromosomal localisation. The sequence is specificallytargeted to, and can hybridize with, a particular location on anindividual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found in, for example, V. McKusick,Mendelian Inheritance in Man (available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

[0044] The differences in the cDNA or genomic sequence between affectedand unaffected individuals can also be determined. If a mutation isobserved in some or all of the affected individuals but not in anynormal individuals, then the mutation is likely to be the causativeagent of the disease.

[0045] The nucleotide sequences of the present invention are alsovaluable for tissue localisation. Such techniques allow thedetermination of expression patterns of the human SC6 polypeptides intissues by detection of the mRNAs that encode them. These techniquesinclude in situ hybridization techniques and nucleotide amplificationtechniques, for example PCR. Such techniques are well known in the art.Results from these studies provide an indication of the normal functionsof the polypeptides in the organism. In addition, comparative studies ofthe normal expression pattern of human SC6 mRNAs with that of mRNAsencoded by a human SC6 gene provide valuable insights into the role ofmutant human SC6 polypeptides, or that of inappropriate expression ofnormal human SC6 polypeptides, in disease. Such inappropriate expressionmay be of a temporal, spatial or simply quantitative nature.

[0046] The polypeptides of the invention or their fragments or analogsthereof, or cells expressing them, can also be used as immunogens toproduce antibodies immunospecific for polypeptides of the presentinvention. The term “immunospecific” means that the antibodies havesubstantially greater affinity for the polypeptides of the inventionthan their affinity for other related polypeptides in the prior art.

[0047] Antibodies generated against polypeptides of the presentinvention may be obtained by administering the polypeptides orepitope-bearing fragments, analogs or cells to an animal, preferably anon-human animal, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature (1975)256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridomatechnique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY,pp.77-96, Alan R. Liss, Inc., 1985).

[0048] Techniques for the production of single chain antibodies, such asthose described in U.S. Pat. No. 4,946,778, can also be adapted toproduce single chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

[0049] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography.

[0050] Antibodies against polypeptides of the present invention may alsobe employed to treat the Diseases, amongst others.

[0051] In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94129458 and WO94/22914.

[0052] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with a polypeptide of the present invention, adequate to produceantibody and/or T cell immune response to protect said animal from theDiseases hereinbefore mentioned, amongst others. Yet another aspect ofthe invention relates to a method of inducing immunological response ina mammal which comprises, delivering a polypeptide of the presentinvention via a vector directing expression of the polynucleotide andcoding for the polypeptide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

[0053] A further aspect of the invention relates to animmunological/vaccine formulation (composition) which, when introducedinto a mammalian host, induces an immunological response in that mammalto a polypeptide of the present invention wherein the compositioncomprises a polypeptide or polynucleotide of the present invention. Thevaccine formulation may further comprise a suitable carrier. Since apolypeptide may be broken down in the stomach, it is preferablyadministered parenterally (for instance, subcutaneous, intramuscular,intravenous, or intradermal injection). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

[0054] Polypeptides of the present invention are responsible for one ormore biological functions, including one or more disease states, inparticular the Diseases hereinbefore mentioned. It is therefore desirousto devise screening methods to identify compounds which stimulate orwhich inhibit the function of the polypeptide. Accordingly, in a furtheraspect, the present invention provides for a method of screeningcompounds to identify those which stimulate or which inhibit thefunction of the polypeptide. In general, agonists or antagonists may beemployed for therapeutic and prophylactic purposes for such Diseases ashereinbefore mentioned. Compounds may be identified from a variety ofsources, for example, cells, cell-free preparations, chemical libraries,and natural product mixtures. Such agonists, antagonists or inhibitorsso-identified may be natural or modified substrates, ligands, receptors,enzymes, etc., as the case may be, of the polypeptide; or may bestructural or functional mimetics thereof (see Coligan et al., CurrentProtocols in Immunology 1(2):Chapter 5 (1991)).

[0055] The screening method may simply measure the binding of acandidate compound to the polypeptide, or to cells or membranes bearingthe polypeptide, or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound.Alternatively, the screening method may involve competition with alabeled competitor. Further, these screening methods may test whetherthe candidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells bearing the polypeptide. Inhibitors of activation aregenerally assayed in the presence of a known agonist and the effect onactivation by the agonist by the presence of the candidate compound isobserved. Constitutively active polypeptides may be employed inscreening methods for inverse agonists or inhibitors, in the absence ofan agonist or inhibitor, by testing whether the candidate compoundresults in inhibition of activation of the polypeptide. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring SC6 activity in the mixture, andcomparing the SC6 activity of the mixture to a standard. Fusionproteins, such as those made from Fc portion and SC6 polypeptide, ashereinbefore described, can also be used for high-throughput screeningassays to identify antagonists for the polypeptide of the presentinvention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); andK. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

[0056] As an example, activity of a neurotransmitter transporter may bedetermined in cells in culture through use of a radiolabelled substrate.After exposure of the cells to the substrate (added to the culturemedium) for a defined length of time, the cells are washed, theircontents extracted by treatment with acid or alkali and the cellularaccumulation of radiolabel determined by scintillation spectrometry. Inthis way, maximal activity, substrate concentration required for halfmaximal activity (Km) and the potency of competing agents (e.g. smallmolecules) can be determined by established methods (see e.g. Clark J Aand Amara S, Mol. Pharmacol. 46 (1994), 550-557). In order to determinewhether competing agents actually inhibit transporter function, it isnecessary to distinguish between agents which block the activity of thetransporter and agents which reduce radiolabelled substrate accumulationin the cells by acting in themselves as substrates for the transporter.This can be assessed by first preloading the cells with radiolabelledsubstrate in the absence of any competing agent. Measurements can thenbe made of the subsequent efflux of radiolabelled substrate from thecells under conditions which favour “reverse” activity of thetransporter (e.g. low extracellular concentrations of Na+ and Cl− ions).An agent which acts as a substrate for the transporter should increasethis efflux, whereas an agent which is a true inhibitor of transporterfunction should decrease it (see e.g. Chen N et al, J. Neurochem. (1998)71, 653-665).

[0057] The polynucleotides, polypeptides and antibodies to thepolypeptide of the present invention may also be used to configurescreening methods for detecting the effect of added compounds on theproduction of mRNA and polypeptide in cells. For example, an ELISA assaymay be constructed for measuring secreted or cell associated levels ofpolypeptide using monoclonal and polyclonal antibodies by standardmethods known in the art. This can be used to discover agents which mayinhibit or enhance the production of polypeptide (also called antagonistor agonist, respectively) from suitably manipulated cells or tissues.

[0058] The polypeptide may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the polypeptide is labeled with aradioactive isotope (for instance, ¹²⁵I), chemically modified (forinstance, biotinylated), or fused to a peptide sequence suitable fordetection or purification, and incubated with a source of the putativereceptor (cells, cell membranes, cell supernatants, tissue extracts,bodily fluids). Other methods include biophysical techniques such assurface plasmon resonance and spectroscopy. These screening methods mayalso be used to identify agonists and antagonists of the polypeptidewhich compete with the binding of the polypeptide to its receptors, ifany. Standard methods for conducting such assays are well understood inthe art.

[0059] Examples of potential polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

[0060] Thus, in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such polypeptides,which comprises:

[0061] (a) a polypeptide of the present invention;

[0062] (b) a recombinant cell expressing a polypeptide of the presentinvention;

[0063] (c) a cell membrane expressing a polypeptide of the presentinvention; or

[0064] (d) antibody to a polypeptide of the present invention;

[0065] which polypeptide is preferably that of SEQ ID NO:2.

[0066] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0067] It will be readily appreciated by the skilled artisan that apolypeptide of the present invention may also be used in a method forthe structure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

[0068] (a) determining in the first instance the three-dimensionalstructure of the polypeptide;

[0069] (b) deducing the three-dimensional structure for the likelyreactive or binding site(s) of an agonist, antagonist or inhibitor;

[0070] (c) synthesing candidate compounds that are predicted to bind toor react with the deduced binding or reactive site; and

[0071] (d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

[0072] It will be further appreciated that this will normally be aniterative process.

[0073] In a further aspect, the present invention provides methods oftreating abnormal conditions such as, for instance, neuropathic pain,other pain, spasticity, myoclonus, epilepsy, stroke, head trauma, spinalcord injury, dystonia, multiple sclerosis, amyotrophic lateralsclerosis, Huntington's Disease or Parkinson's Disease, related toeither an excess of, or an under-expression of, SC6 polypeptideactivity.

[0074] If the activity of the polypeptide is in excess, severalapproaches are available. One approach comprises administering to asubject in need thereof an inhibitor compound (antagonist) ashereinabove described, optionally in combination with a pharmaceuticallyacceptable carrier, in an amount effective to inhibit the function ofthe polypeptide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide may be administered. Typical examples of suchcompetitors include fragments of the SC6 polypeptide.

[0075] In still another approach, expression of the gene encodingendogenous SC6 polypeptide can be inhibited using expression blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or externally administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides whichform triple helices (“triplexes”) with the gene can be supplied (see,for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et al.,Science (1988) 241:456; Dervan et al., Science (1991) 251:1360). Theseoligomers can be administered per se or the relevant oligomers can beexpressed in vivo. Synthetic antisense or triplex oligonucleotides maycomprise modified bases or modified backbones. Examples of the latterinclude methylphosphonate, phosphorothioate or peptide nucleic acidbackbones. Such backbones are incorporated in the antisense or triplexoligonucleotide in order to provide protection from degradation bynucleases and are well known in the art. Antisense and triplex moleculessynthesised with these or other modified backbones also form part of thepresent invention.

[0076] In addition, expression of the human SC6 polypeptide may beprevented by using ribozymes specific to the human SC6 mRNA sequence.Ribozymes are catalytically active RNAs that can be natural or synthetic(see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4),527-33.) Synthetic ribozymes can be designed to specifically cleavehuman SC6 mRNAs at selected positions thereby preventing translation ofthe human SC6 mRNAs into functional polypeptide. Ribozymes may besynthesised with a natural ribose phosphate backbone and natural bases,as normally found in RNA molecules. Alternatively the ribosymes may besynthesised with non-natural backbones to provide protection fromribonuclease degradation, for example, 2′-O-methyl RNA, and may containmodified bases.

[0077] For treating abnormal conditions related to an under-expressionof SC6 and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates a polypeptide of thepresent invention, i.e., an agonist as described above, in combinationwith a pharmaceutically acceptable carrier, to thereby alleviate theabnormal condition. Alternatively, gene therapy may be employed toeffect the endogenous production of SC6 by the relevant cells in thesubject. For example, a polynucleotide of the invention may beengineered for expression in a replication defective retroviral vector,as discussed above. The retroviral expression construct may then beisolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding a polypeptide of thepresent invention such that the packaging cell now produces infectiousviral particles containing the gene of interest. These producer cellsmay be administered to a subject for engineering cells in vivo andexpression of the polypeptide in vivo. For an overview of gene therapy,see Chapter 20, Gene Therapy and other Molecular Genetic-basedTherapeutic Approaches, (and references cited therein) in HumanMolecular Genetics, T Strachan and A P Read, BIOS Scientific PublishersLtd (1996). Another approach is to administer a therapeutic amount of apolypeptide of the present invention in combination with a suitablepharmaceutical carrier.

[0078] In a further aspect, the present invention provides forpharmaceutical compositions comprising a therapeutically effectiveamount of a polypeptide, such as the soluble form of a polypeptide ofthe present invention, agonist/antagonist peptide or small moleculecompound, in combination with a pharmaceutically acceptable carrier orexcipient. Such carriers include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0079] The composition will be adapted to the route of administration,for instance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

[0080] The dosage range required depends on the choice of peptide orother compounds of the present invention, the route of administration,the nature of the formulation, the nature of the subject's condition,and the judgment of the attending practitioner. Suitable dosages,however, are in the range of 0.1-100 μg/kg of subject. Wide variationsin the needed dosage, however, are to be expected in view of the varietyof compounds available and the differing efficiencies of various routesof administration. For example, oral administration would be expected torequire higher dosages than administration by intravenous injection.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

[0081] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy” as described above. Thus, for example, cells from a subject maybe engineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

[0082] Polynucleotide and polypeptide sequences form a valuableinformation resource with which to identify further sequences of similarhomology. This is most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data to search asequence database using well known searching tools, such as those in theGCG and Lasergene software packages. Accordingly, in a further aspect,the present invention provides for a computer readable medium havingstored thereon a polynucleotide comprising the sequence of SEQ ID NO:1and/or a polypeptide sequence encoded thereby.

[0083] The following definitions are provided to facilitateunderstanding of certain terms used frequently hereinbefore.

[0084] “Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

[0085] “Isolated” means altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein.

[0086] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0087] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as post-translational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance,PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York, 1983; Seifter et al., “Analysis for proteinmodifications and nonprotein cofactors”, Meth Enzymol (1990) 182:626-646and Rattan et al., “Protein Synthesis: Post-translational Modificationsand Aging”, Ann NY Acad Sci (1992) 663:48-62).

[0088] “Variant” refers to a polynucleotide or polypeptide that differsfrom a reference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

[0089] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

[0090] Preferred parameters for polypeptide sequence comparison includethe following:

[0091] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0092] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0093] Gap Penalty: 12

[0094] Gap Length Penalty: 4

[0095] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0096] Preferred parameters for polynucleotide comparison include thefollowing:

[0097] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0098] Comparison matrix: matches=+10, mismatch=0

[0099] Gap Penalty: 50

[0100] Gap Length Penalty: 3

[0101] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0102] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:1,that is be 100% identical, or it may include up to a certain integernumber of nucleotide alterations as compared to the reference sequence.Such alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in SEQ ID NO:1 by the numerical percent of the respectivepercent identity (divided by 100) and subtracting that product from saidtotal number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) •y),

[0103] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, and y is, for instance,0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc., and wherein any non-integer product of x_(n) and y is rounded downto the nearest integer prior to subtracting it from x_(n). Alterationsof a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 maycreate nonsense, missense or frameshift mutations in this codingsequence and thereby alter the polypeptide encoded by the polynucleotidefollowing such alterations.

[0104] Similarly, a polypeptide sequence of the present invention may beidentical to the reference sequence of SEQ ID NO:2, that is be 100%identical, or it may include up to a certain integer number of aminoacid alterations as compared to the reference sequence such that the %identity is less than 100%. Such alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of amino acidalterations for a given % identity is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the numerical percent ofthe respective percent identity (divided by 100) and then subtractingthat product from said total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) •y),

[0105] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, and y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein anynon-integer product of x_(a) and y is rounded down to the nearestinteger prior to subtracting it from x_(a).

[0106] “Homolog” is a generic term used in the art to indicate apolynucleotide or polypeptide sequence possessing a high degree ofsequence relatedness to a subject sequence. Such relatedness may bequantified by determining the degree of identity and/or similaritybetween the sequences being compared as hereinbefore described. Fallingwithin this generic term are the terms “ortholog”, meaning apolynucleotide or polypeptide that is the functional equivalent of apolynucleotide or polypeptide in another species, and “paralog” meaninga functionally similar sequence when considered within the same species.

[0107] “Fusion protein” refers to a protein encoded by two, oftenunrelated, fused genes or fragments thereof. In one example, EP-A-0 464discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232 262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

[0108] All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

EXAMPLE 1 Cloning

[0109] The nucleotide sequence 256 to 2649 of SEQ ID NO:1 was obtainedby PCR using gene specific primers, SEQ ID NO:3 (5′ primer) and SEQ IDNO:4 (3′ primer), to amplify up the full length clone. The template usedfor this PCR was 1st strand cDNA, produced using AMV Reversetranscriptase and an oligo(dT) primer, on a 500 ng poly A+Spinal CordRNA (Clontech). The reverse transcriptase reaction was carried out usingthe Promega RT system. Reaction conditions were: 1×RT buffer, 1 mMdNTPs, 5 mM Mg²⁺, 10 units RNasin, 7.5 units AMV reverse transcriptase,0.25 ug oligo(dT)15 Primer, 500 ng RNA template. The oligo(dT) primerwas added to the polyA+RNA and the reaction incubated for 70° C. for 10minutes, then placed on ice. The reaction was then incubated at 42° C.for 60 minutes, followed by 99° C. for 5 minutes and then onto ice. Thereaction product was diluted 1:10 for use in the PCR reaction.

[0110] The PCR reaction was carried out in 50 ul volume: 1×KlentaqBuffer, 0.2 mM dNTPs, 100 nM 5′Primer (SEQ ID NO:3), 100 nM 3′Primer(SEQ ID NO:4), 5 ul Spinal cord 1st strand cDNA (above), 1×KlenTaqpolymerase mix. Cycling was at 95° C. for 2 minutes followed by 35cycles of: 95° C. for 30 sec; 62° C. for 30 sec and 68° C. for 4 min.This was followed by a 10 minute hold at 68° C., then to 4° C.

[0111] Also included in Seq ID NO:1 are 5′ and 3′ untranslated (UTR)sequences. These were obtained separately. The 5′UTR (nucleotide 1 to255) was obtained from an independent PCR on a Marathon cerebellum cDNAtemplate, using 5′RACE PCR conditions (as recommended by themanufacturer) with gene specific primers, SEQ ID NO:5 (first-roundprimer) and SEQ ID NO:6 (nesting primer), together with Marathon adapterprimers. The 3′ UTR (nucleotide 2650 to 2863) was obtained by sequencingof Image clone No:745336; Soares NHT normalised human testis.

EXAMPLE 2 Mammalian Cell Expression

[0112] The transporter of the present invention was expressedtransiently in human embryonic kidney 293 (HEK-293) cells. To maximiseexpression, typically 5′ and 3′ untranslated regions (UTRs) are removedfrom the transporter cDNA prior to transfection. Cells were transientlytransfected using lipofectin. 24-48 hours after transfection, glycinetransport activity was assayed as described in Example 2.

EXAMPLE 3 Glycine Uptake Assay

[0113] Suspensions of cells transiently transfected as described inExample 1 were pipetted into polylysine-colated 96 well plates (50,000cells per well). 4-24 hours later, the wells were washed with standardKrebs/HEPES buffer (KHB) at 37° C. After addition of further KHB to thewells, the plates were incubated at 37° C. for 10 min. KHB containingappropriate concentrations of a potential inhibitor was then added tothe wells, together with 1 μM [3H]-glycine. After incubation at 37° C.for 2-60 min, the wells were washed with ice-cold KHB. The cells werethen solubilised in NaOH, and the amount of radioactivity presentdetermined using a scintillation counter.

[0114] An example of the time course of [3H]-glycine uptake in cellstransfected with SC6 is shown in FIG. 1. This [3H]-glycine uptake isalmost totally blocked by inclusion of non-radioactive glycine (1 mM) inthe assay, but is not significantly affected by sarcosine (1 mM). Thislack of effect of sarcosine (a blocker of uptake via the GlyT-1transporter), together with the fact that all assays were performed inthe presence of alanine (5 mM) to block uptake via neutral amino acidtransporter component of uptake, indicates that the [3H]-glycine uptakewas mediated by the GlyT-2 transporter (Liu Q R et al, J. Biol. Chem.(1993) 268, 220802-22808).

[0115] [3H]-glycine uptake as a function of the total concentration ofglycine added to cells transfected with SC6 is shown in FIG. 2. Theresults indicate a Km value of 58 nM and a Vmax value of 1130 pmoles/mgprotein/min. SEQUENCE INFORMATION SEQ ID NO:1CCCTCCCGCTGGAGTGACAACTGGCCAGCATACTCTAGGCTGTTGTCCCTTTAAAACTTGAATCCAAGGGGGTAATGATTTATCAAACTTGTATTATCAAGAAAATGTCAAACCAAGGGCACCTTGCTTTGCACTGACGCAAACCCGGCCTTTCCCAAGGAGATATAGAAAGCGCCTCTCCTGCCTGAGCCAAACCCAGTCTTGTCAATAGCGGGTTTCACCCTCCACTAGTTCAGTCTGTTGCCTGTGTCAGACATGGATTGCAGTGCTCCCAAGGAAATGAATAAACTGCCAGCCAACAGCCCGGAGGCGGCGGCGGCGCAGGGCCACCCGGATGGCCCATGCGCTCCCAGGACGAGCCCGGAGCAGGAGCTTCCCGCGGCCGCCGCCCCGCCGCCGCCACGTGTGCCCAGGTCCGCTTCCACCGGCGCCCAAACTTTCCAGTCAGCGGACGCGCGAGCCTGCGAGGCTGAGCGGCCAGGAGTGGGGTCTTGCAAACTCAGTAGCCCGCGQGCGCAGGCGGCCTCTGCAGCTCTGCGGGACTTGAGAGAGGCGCAAGGCGCGCAGGCCTCGCCCCCTCCCGGGAGCTCCGGGCCCGGCAACGCGCTGCACTGTAAGATCCCTTCTCTGCGAGGCCCGGAGGGGGATGCGAACGTGAGTGTGGGCAAGGGCACCCTGGAGCGGAACAATACCCCTGTTGTGGGCTGGGTGAACATGAGCCAGAGCACCGTGGTGCTGGGCACGGATGGAATCACGTCCGTGCTCCCGGGCAGCGTGGCCACCGTTGCCACCCAGGAGGACGAGCGAGGGGATGAGAATAAGGCCCGAGGGAACTGGTCCAGCAAACTGGACTTCATCCTGTCCATGGTGGGGTACGCAGTGGGGCTGGGCAATGTCTGGAGGTTTCCCTACCTGGCCTTCCAGAACGGGGGAGGTGCTTTCCTCATCCCTTACCTGATGATGCTGGCTCTGGCTGGATTACCCATCTTCTTCTTGGAGGTGTCGCTGGGCCAGTTTGCCAGCCAGGGACCAGTGTCTGTGTGGAAGGCCATCCCAGCTCTACAAGGCTGTGGCATCGCGATGCTGATCATCTCTGTCCTAATAGCCATATACTACAATGTGATTATTTGCTATACACTTTTCTACCTGTTTGCCTCCTTTGTGTCTGTACTACCCTGGGGCTCCTGCAACAACCCTTGGAATACGCCAGAATGCAAAGATAAAACCAAACTTTTATTAGATTCCTGTGTTATCAGTGACCATCCCAAAATACAGATCAAGAACTCGACTTTCTGCATGACCGCTTATCCCAACGTGACAATGGTTAATTTCACCAGCCAGGCCAATAAGACATTTGTCAGTGGAAGTGAAGAGTACTTCAAGTACTTTGTGCTGAAGATTTCTGCAGGGATTGAATATCCTGGCGAGATCAGGTGGCCACTAGCTCTCTGCCTCTTCCTGGCTTGGQTCATTGTGTATGCATCGTTGGCTAAAGGAATCAAGACTTCAGGAAAAGTGGTGTACTTCACGGCCACGTTCCCGTATGTCGTACTCGTGATCCTCCTCATCCGAGGAGTCACCCTGCCTGGAGCTGGAGCTGGGATCTGGTACTTCATCACACCCAAGTGGGAGAAACTCACGAATGCCACGGTGTGGAAAGATGCTGCCACTCAGATTTTCTTCTCTTTATCTGCTGCATGGGGAGGCCTGATCACTCTCTCTTCTTACAACAAATTCCACAACAACTGCTACAGGGACACTCTAATTGTCACCTGCACCAACAGTGCCACAAGCATCTTTGCCGGCTTCGTCATCTTCTCCGTTATCGGCTTCATGGCCAATGAACGCAAAGTCAACATTGAGAATGTGGCAGACCAAGGGCCAGGCATTGCATTTGTGGTTTACCCGGAAGCCTTAACCAGGCTGCCTCTCTCTCCGTTCTGGCCCATCATCTTTTTCCTGATGCTCCTCACTCTTGGACTTGACACTATGTTTGCCACCATCGAGACCATAGTGACCTCCATCTCAGACGAGTTTCCCAAGTACCTACGCACACACAAGCCAGTGTTTACTCTGGGCTGCTGCATTTGTTTCTTCATCATGGGTTTTCCAATGATCACTCAGGGTGGAATTTACATGTTTCAGCTTGTGGACACCTATGCTGCCTCCTATGCCCTTGTCATCATTGCCATTTTTGAGCTCGTGGGGATCTCTTATGTGTATGGCTTGCAAAGATTCTGTGAAGATATAGAGATGATGATTGGATTCCAGCCTAACATCTTCTGGAAAGTCTGCTGGGCATTTGTAACCCCAACCATTTTAACCTTTATCCTTTGCTTCAGCTTTTACCAGTGGGAGCCCATGACCTATGGCTCTTACCGCTATCCTAACTGGTCCATGGTGCTCGGATGGCTAATGCTCGCCTGTTCCGTCATCTGGATCCCAATTATGTTTGTGATAAAAATGCATCTGGCCCCTGGAAGATTTATTGAGAGGCTGAAGTTGGCGTGCTCGCCACAGCCGGACTGGGGCCCATTCTTAGCTCAACACCGCGGGGACGCTTACAAGAACATGATCGACCCCTTGGGAACCTCTTCCTTGGGACTCAAACTGCCAGTGAAGGATTTGGAACTGGGCACTCAGTGCTAGTCCAGTGGTGTGGGATGGTCCAGACTTGATCCTGTTTTTCCTCTCTGCCTCCTCCTAATGTTTTCCATAGCTCTCCTCCCATTTTTCTTCATCTTTCTTCCTACATCTTGGTTCACATCCACGCATGAGAGTGATTATGTAGAAAAGTAGGCATAGTGTCGCATGCTGCAGTAAAGAGCTACATAGACCACCTGAAAAAAA AAAAAAAAAAAAA SEQ IDNO:2 MDCSAPKEMNKLPANSPEAAAAQGHPDGPCAPRTSPEQELPAAAAPPPPRVPRSASTGAQTFQSADARACEAERPGVGSCKLSSPRAQAASAALRDLREAQGAQASPPPGSSGPGNALHCKIPSLRGPEGDANVSVGKGTLERNNTPVVGWVNMSQSTVVLGTDGITSVLPGSVATVATQEDERGDENKARGNWSSKLDFILSMVGYAVGLGNVWRFPYLAFQNGGGAFLIPYLMMLALAGLPIFFLEVSLGQFASQGPVSVWKAIPALQGCGIAMLIISVLIAIYYNVIICYTLFYLFASFVSVLPWGSCNNPWNTPECKDKTKLLLDSCVISDHPKIQIKNSTFCMTAYPNVTMVNFTSQANKTFVSGSEEYFKYFVLKISAGIEYPGEIRWPLALCLFLAWVIVYASLAKGIKTSGKVVYFTATFPYVVLVILLIRGVTLPGAGAGIWYFITPKWEKLTNATVWKDAATQIFFSLSAAWGGLITLSSYNKFHNNCYRDTLIVTCTNSATSIFAGFVIFSVIGFMANERKVNIENVADQGPGIAFVVYPEALTRLPLSPFWAIIFFLMLLTLGLDTMFATIETIVTSISDEFPKYLRTHKPVFTLGCCICFFIMGFPMITQGGIYMFQLVDTYAASYALVIIAIFELVGISYVYGLQRFCEDIEMMIGFQPNIFWKVCWAFVTPTILTFILCFSFYQWEPMTYGSYRYPNWSMVLGWLMLACSVTWTPIMFVTKMHLAPGRFIERLKLACSPQPDWGPFLAQHRGERYKNMTDPLGTSSLGLKIPVKDLELGTQC SEQ ID NO:3 GCC ACC ATGGAT TGC AGT GCT CCC AAG GA SEQ ID NO:4 GGA CTA GCA CTG AGT GCC CAG TTC CSEQ ID NO:5 CTC TCA AGT CCC GCA GAG CTG CAG SEQ ID NO:6 GGC TAC TGA GTTTGC AAG AC

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidsequence selected from the group having at least: (a) 97% identity; (b)98% identity; or (c) 99% identity to the amino acid sequence of SEQ IDNO:2 over the entire length of SEQ ID NO:2; (ii) an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:2 or (iii) an isolatedpolypeptide which is the amino acid sequence of SEQ ID NO:2.
 2. Anisolated polynucleotide selected from the group consisting of: (i) anisolated polynucleotide comprising a nucleotide sequence encoding apolypeptide that has at least (a) 97% identity; (b) 98% identity; or (c)99% identity to the amino acid sequence of SEQ ID NO:2, over the entirelength of SEQ ID NO:2; (ii) an isolated polynucleotide comprising anucleotide sequence that has at least: (a) 90% identity; or (b) 95%identity; over its entire length to a nucleotide sequence encoding thepolypeptide of SEQ ID NO:2; (iii) an isolated polynucleotide comprisinga nucleotide sequence which has at least: (a) 90% identity; or (b) 95%identity; to that of SEQ ID NO:1 over the entire length of SEQ ID NO:1;(iv) an isolated polynucleotide comprising a nucleotide sequenceencoding the polypeptide of SEQ ID NO:2; (v) an isolated polynucleotidewhich is the polynucleotide of SEQ ID NO:1; or (vi) an isolatedpolynucleotide obtainable by screening an appropriate library understringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or a fragment thereof; or a nucleotide sequencecomplementary to said isolated polynucleotide.
 3. An antibodyimmunospecific for the polypeptide of claim 1 .
 4. A method for thetreatment of a subject: (i) in need of enhanced activity or expressionof the polypeptide of claim 1 comprising: (a) administering to thesubject a therapeutically effective amount of an agonist to saidpolypeptide; and/or (b) providing to the subject an isolatedpolynucleotide comprising a nucleotide sequence encoding saidpolypeptide in a form so as to effect production of said polypeptideactivity in vivo; or (ii) having need to inhibit activity or expressionof the polypeptide of claim 1 comprising: (a) administering to thesubject a therapeutically effective amount of an antagonist to saidpolypeptide; and/or (b) administering to the subject a nucleic acidmolecule that inhibits the expression of a nucleotide sequence encodingsaid polypeptide; and/or (c) administering to the subject atherapeutically effective amount of a polypeptide that competes withsaid polypeptide for its ligand, substrate, or receptor.
 5. A processfor diagnosing a disease or a susceptibility to a disease in a subjectrelated to expression or activity of the polypeptide of claim 1 in asubject comprising: (a) determining the presence or absence of amutation in the nucleotide sequence encoding said polypeptide in thegenome of said subject; and/or (b) analyzing for the presence or amountof said polypeptide expression in a sample derived from said subject. 6.A method for screening to identify compounds which stimulate or whichinhibit the function of the polypeptide of claim 1 which comprises amethod selected from the group consisting of: (a) measuring the bindingof a candidate compound to the polypeptide (or to the cells or membranesbearing the polypeptide) or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound; (b)measuring the binding of a candidate compound to the polypeptide (or tothe cells or membranes bearing the polypeptide) or a fusion proteinthereof in the presence of a labeled competitor; (c) testing whether thecandidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells or cell membranes bearing the polypeptide; (d) mixing acandidate compound with a solution containing a polypeptide of claim 1 ,to form a mixture, measuring activity of the polypeptide in the mixture,and comparing the activity of the mixture to a standard; or (e)detecting the effect of a candidate compound on the production of mRNAencoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay.
 7. An agonist or an antagonist of thepolypeptide of claim 1 .
 8. An expression system comprising apolynucleotide capable of producing a polypeptide of claim 1 when saidexpression system is present in a compatible host cell.
 9. A process forproducing a recombinant host cell comprising transforming ortransfecting a cell with the expression system of claim 8 such that thehost cell, under appropriate culture conditions, produces a polypeptidecomprising an amino acid sequence having at least 97% identity to theamino acid sequence of SEQ ID NO:2 over the entire length of SEQ IDNO:2.
 10. A recombinant host cell produced by the process of claim 9 .11. A membrane of a recombinant host cell of claim 10 expressing apolypeptide comprising an amino acid sequence having at least 97%identity to the amino acid sequence of SEQ ID NO:2 over the entirelength of SEQ ID NO:2.
 12. A process for producing a polypeptidecomprising culturing a host cell of claim 10 under conditions sufficientfor the production of said polypeptide and recovering the polypeptidefrom the culture.